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Figure 1. Proximity ligation assay. The proximity ligation assay (PLA) workflow consists of four main steps: (1) Antibodies targeting adjacent epitopes on the same Aspergillus -specific antigen are biotinylated and attached to different streptavidin-linked oligodeoxynucleotides, creating 'proximity probes'. (2) The two probes are combined and incubated with samples, allowing antibodies to bind to their epitopes. The reaction mixture also contains a single-stranded oligonucleotide that is complementary to the ends of the two oligonucleotides. If antigen is present, the oligonucleotides are linked.
(3) A ligation reaction seals the gap. (4) This generates a template that can be amplified by PCR. (5) The amplification plot shows a PLA carried out using JF5 antibody.
(Figure omitted. See article PDF.)
Figure 2. Microscopic views of different fungi reveals the similarities between different Aspergillus species. (A) Aspergillus niger , (B) A. candidus , (C) A. flavus , (D) A. fumigatus. Reprinted with permission from Melvyn Eydmann and Tim Linehan (Microbiology Department, Royal London Hospital, UK).
(Figure omitted. See article PDF.)
Figure 3. Phylogenetic tree based on fungal 18S ribosomal DNA sequences using the unweighted pair group method with arithmetic mean algorithm from the CLC Sequence Viewer 6.8.2 (CLC bio A/S, Denmark). Several Aspergillus fumigatus strains cluster together at different locations, all Candida cluster together and Penicillium chrysogenum is closely related to A. candidus . The branch annotations are the bootstrap values obtained from 1000 bootstrap tests.
(Figure omitted. See article PDF.)
Figure 4. Antibody-based detection of Aspergillus antigens. (A) Colorimetric detection of GM. Key: clear: GM absent; yellow: GM present. (B) Lateral flow device showing a positive result for Aspergillus detection, indicated by the two bands, one of which serves as a positive assay control, and the other being specific for the presence of antigen recognized by the monoclonal antibody JF5.
(Figure omitted. See article PDF.)
Figure 5. Colourimetric detection of siderophore production by Aspergillus fumigatus under iron-free and replete conditions, respectively. Maximum siderophore production (800 μg/ml) was evident when Aspergillus fumigatus was cultured in the absence of iron. Key: blue: siderophores absent; orange/pink: siderophores present.
(Figure omitted. See article PDF.)
Figure 6. Collection of exhaled breath condensate using a condensation device designed to exclude gross salivary contamination. Patients sitting comfortably breathe at tidal volumes into a mouthpiece attached...